The long-term goal of this project is to elucidate the mechanism of Ca2+ -mediated cell death in cardiomyopathy. It is our hypothesis that altered Ca2+ homeostasis and unbalanced signal transduction can lead to mitochondrial dysfunction and cell death. We propose: 1. To study the regulation of mitochondrial Ca2+ (Cam) by the mitochondrial Na/Ca exchanger (NCE) and the permeability transition (MPT) pore in isolated organelle or cultured myocytes. 2. To study the effect of proapoptotic signal transduction on mitochondria by comparing the sensitivity to Ca2+ mediated death and mitochondrial function (Cam load, delta psi, NCE activity and MPT) in cells that overexpress the stress-activated kinase JNK with cells that expressing mutant inactive-JNK. 3. To study the development of cardiomyopathy in the Syrian hamster. The development of lesion in the myopathic heart will be followed at pre-necrotic- (1 mo), necrotic- (2 mo.) and post necrotic- (3 mo.) stage. The changes in the heart will be correlated with studies in isolated myocytes and mitochondria. The parameters to be monitored are mitochondrial Cam load, abnormal NCE activity, JNK/ERK activation, and susceptibility to Ca2+ -induced permeability transition. Intervention of cardiomyopathy will be carried out by diltiazem treatment, and heart tissue or mitochondria examined for the prevention of lesion development and for the reversal of abnormal Ca2+ homeostasis and NCE activity. 4. To study the effects of sarcolemmal Na/Ca exchanger (NCX1) overexpression on mitochondrial function and cell injury in transgenic mice. The mechanism for the gender-specific increase in susceptibility to Ca2+ -mediated injury, and the upregulation of mitochondrial NCE activity in association with NCX 1 overexpression will be tested. The direct effect of estrogen (E2) on the activation of growth promoting kinase ERK, and on Ca2+ homeostasis during metabolic stress will also be examined in normal rat myocytes and compared to transgenic myocytes. These studies are designed to advance a poorly understood topic, that is the role of mitochondrial calcium homeostasis and signal transduction in cell survival and cardiomyopathy. We will examine new aspects in the mechanism of cell death and expect the results to be highly relevant to our understanding of gender- specific myocyte function in normal and disease states.